Shifts in the selectivity filter dynamics cause modal gating in K+ channels

Abstract

Spontaneous activity shifts at constant experimental conditions represent a widespread regulatory mechanism in ion channels. The molecular origins of these modal gating shifts are poorly understood. In the K⁺ channel KcsA, a multitude of fast activity shifts that emulate the native modal gating behaviour can be triggered by point-mutations in the hydrogen bonding network that controls the selectivity filter. Using solid-state NMR and molecular dynamics simulations in a variety of KcsA mutants, here we show that modal gating shifts in K⁺ channels are associated with important changes in the channel dynamics that strongly perturb the selectivity filter equilibrium conformation. Furthermore, our study reveals a drastically different motional and conformational selectivity filter landscape in a mutant that mimics voltage-gated K⁺ channels, which provides a foundation for an improved understanding of eukaryotic K⁺ channels. Altogether, our results provide a high-resolution perspective on some of the complex functional behaviour of K⁺ channels.

Fig. 1

E71X point-mutations cause large conformational changes in the KcsA selectivity filter. a The selectivity filter of K⁺ channel KcsA (1K4C) is regulated by a hydrogen bond network with the triad W67–E71–D80 at the centre. b W67 and D80 are highly conserved, while E71 is commonly replaced by a nonpolar valine or isoleucine in Kv channels. c 2D NH ssNMR spectra of WT KcsA (cyan) and mutant E71A (red) acquired in membranes. Arrows indicate major signal shifts of key residues. Residues L41–W87 are annotated in the E71A spectrum and highlighted in red on the X-ray structure. d Chemical shift perturbations (CSPs) of E71A (red), E71I (blue), and E71Q (orange) in reference to WT KcsA. Combined HN CSPs (left) of amino-protons and backbone-nitrogens and (right) Cα CSPs. The strongest NH CSPs in E71A are highlighted in c. Source data are provided as a Source Data file. e 2D NH (upper panel) and 2D CC spectra (lower) showing large CSPs of key residues W67, V76, Y78, and D80 in E71A (red), E71I (blue), and E71Q (orange) relative to WT KcsA (cyan)

Fig. 2

Loss of the E71–D80 interaction causes extended rearrangements behind the filter. a Zoom into (left) 2D CC ssNMR spectra of WT KcsA (cyan), E71A (red), E71I (blue), and E71Q (orange) showing the D80 side chain CSPs. E71Q (right) mimics the E71–D80 interaction, which is lost in E71A and E71I. The D80 side chain CSPs are large in E71A and E71I, while they are small in E71Q. b Structural representation (1K4C) of the stabilisation of filter residues T74–G77 by hydrogen bonds with V70 and E71 of the pore helix. c The E71I turret is disordered, which causes signals to disappear or split. Spectral zooms are show for WT KcsA (cyan) and E71I (blue). d Overlay of 2D NH spectra showing a strong CSP for V70 of the pore helix. e Overlay of 2D CC spectra, showing CSPs of the functionally critical T74 side chain in E71X mutants. f The large T74 CSP in E71I (pH 7.4, 100 mM K⁺) is reminiscent of the inactivated filter in WT KcsA (pH 4, 0 mM K⁺)

Fig. 3

E71X point-mutations strongly change the selectivity filter dynamics. a ¹⁵N rotating-frame ssNMR relaxation rates (R_1rho) that report on slow molecular motions in WT KcsA (cyan), E71A (red), E71I (blue), and E71Q (orange) measured at 700 MHz and 58 kHz MAS. The error bars show the standard error of the fit. Source data are provided as a Source Data file. b R_1rho signal decay curves for selected filter residues. Symbols mark data points and lines represent best fits. c Plots of the differences in the dynamics between E71X mutants and WT KcsA. d Illustration of the site-resolved selectivity filter dynamics. The size of the magenta spheres represents the R_1rho relaxation rates

Fig. 4

High-resolution analysis of the size of the water cavity behind the filter. a Zoom into 2D NH ssNMR spectra acquired in (upper panel) protonated and (lower) deuterated buffers of WT KcsA (cyan), E71I (blue), E71A (red), and E71Q (orange). b Cross-sections from 2D NH spectra of WT KcsA, E71I, and E71A measured in protonated (continuous lines) and deuterated (dashed) buffers. For Y78 in WT KcsA, cross-sections were extracted from 3D CANH experiments to resolve spectral overlap. Signals are normalised (see Methods). c 2D NH spectra of E71A in protonated (red) and deuterated (grey) buffers showing the fast exchange of Y78, implying a larger water cavity. d Illustrations of the ssNMR-derived water cavity size: in WT KcsA, the cavity is limited to G79-L81, and Y78 is exchange-protected. The cavity widens in E71I, strongly widens in E71A, and is absent in E71Q. Blue and brown spheres represent water-exposed and shielded amino-protons, respectively

Fig. 5

E71X mutations shift the equilibrium between inwards and outwards filter states. a Dihedral angle distribution of filter residues of WT (cyan), E71A (red), E71I (blue), and E71Q (orange) derived from 1-μs-long MD simulations. Characteristic angular spaces for inwards conformations, with the carbonyl group oriented towards the filter pore, and outwards states are highlighted. b Comparison of V76CO CSPs derived from experiments (black bars) and back-calculated from MD simulations (grey bars). Source data are provided as a Source Data file. c Histogram of back-calculated chemical shifts for V76CO and Y78Cα of WT KcsA (cyan) and E71I (blue). The V76CO (left) inwards state is stabilised in E71I, leading to higher V76CO chemical shifts, while the Y78CO (right) inwards state is destabilised in E71I, leading to lower Y78Cα chemical shifts. d Representative MD snapshots of WT KcsA and E71I showing inwards and outwards states of V76CO and Y78CO. The chemical shifts (in magenta) of V76CO and Y78Cα strongly differ between inwards and outwards conformations. e Illustration of the stabilisation of the V76CO inwards state in E71A and E71I, and the destabilisation in E71Q

Fig. 6

The functionally critical W67–D80 interaction is destabilised in E71I KcsA. a WT KcsA MD simulation: the tight interaction with E71 locks the D80 side chain in a down configuration that enables hydrogen bonding with W67 (snapshot after 270 ns). b E71A simulation: the down conformation prevails, enabling the W67–D80 interaction, which stabilises the filter entrance (snapshot after 600 ns). c E71I simulation: I71 impedes the W67–D80 interaction which destabilises the filter entrance. D80 engages in interactions with Y82 (from a neighbouring channel subunit) and R64 (snapshot after 600 ns). d Longitudinal relaxation times (¹⁵N T₁) that report on fast motion of the W67 side chain for E71A (red circles) and E71I (blue squares), measured at 950 MHz and 60 kHz MAS. The error shows the signal-to-noise ratio for W67Nε at a given data point. Source data are provided as a Source Data file. e (left) The W67–D80 interaction is maintained in E71A; (right) I71 hinders the W67–D80 hydrogen bond, which entails increased dynamics at the pore mouth